Reelin rescues cognitive function

ABSTRACT

Disclosed are methods of influencing, and enhancing, cognitive function by increasing, and/or preventing interference with, Reelin levels as well as Reelin signaling. Cognitive function is improved, in a subject in need thereof, by administering a therapeutically effective amount of Reelin, a Reelin-specific modulator or an agonist of a lipoprotein receptor to the subject. The lipoprotein receptor can be selected from candidates such as ApoER2 and VLDLR. As disclosed herein, agonists of the lipoprotein receptor for use with the inventive method include APC, Sep and Fc-RAP. In addition to administering exogenous Reelin, a Reelin-specific modulator, such as a recombinant Reelin fragment, can be used to increase Reelin levels and/or signaling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 13/206,174, filed on Aug. 9, 2011, which is a continuation ofInternational Application, Serial Number PCT/US2010/023615 filed Feb. 9,2010, which claims priority to U.S. Provisional Patent Application No.61/150,890, filed Feb. 9, 2009; the contents of each of which are hereinincorporated by reference.

GOVERNMENT INTEREST STATEMENT

This invention was made with Government support under Grant Number R01NS043408 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The lipoprotein receptor signaling system is known to play a significantrole in the adult CNS such as cholesterol homeostasis, clearance ofextracellular proteins, modulating memory formation, synaptictransmission, plasticity and maturation through the activation ofnumerous signal transduction pathways. Importantly, the lipoproteinreceptor ligand apolipoprotein E (apoE) is one of the best validatedrisk factors for late-onset, sporadic Alzheimer's disease (AD) (Hoe H S,Harris D C, Rebeck G W. Multiple pathways of apolipoprotein E signalingin primary neurons. J Neurochem 2005; 93:145-155; Hoe H S, Freeman J,Rebeck G W. Apolipoprotein E decreases tau kinases and phospho-taulevels in primary neurons. Mol Neurodegener 2006, 1:18; Hoe H S,Pocivaysek A, Chakraborty G, et al. Apolipoprotein E receptor 2interactions with the N-methyl-Daspartate receptor. J Biol Chem 2006,281:3425-3431). Moreover, the extracellular matrix protein reelin canbind to both lipoprotein receptors and amyloid precursor protein (APP)and is known to be associated with Aβ plaques in a number of AD mousemodels (Chin J, Massaro C M, Palop J J, et al. Reelin depletion in theentorhinal cortex of human amyloid precursor protein transgenic mice andhumans with Alzheimer's disease. J Neurosci 2007, 27:2727-2733; HoareauC, Borrell V, Soriano E, Krebs M O, Prochiantz A, Allinquant B. Amyloidprecursor protein cytoplasmic domain antagonizes reelin neuriteoutgrowth inhibition of hippocampal neurons. Neurobiol Aging 2008,29:542-553; Hoe H S, Tran T S, Matsuoka Y, Howell B W, Rebeck G W. DAB1and Reelin effects on amyloid precursor protein and ApoE receptor 2trafficking and processing. J Biol Chem 2006, 281:35176-35185; andMiettinen R, Riedel A, Kalesnykas G, et al. Reelin-immunoreactivity inthe hippocampal formation of 9-month-old wildtype mouse: effects ofAPP/PS1 genotype and ovariectomy. J Chem Neuroanat 2005, 30:105-1180).Aβ accumulation can influence reelin signaling and lipoprotein receptorfunction, thereby promoting AD pathogenesis and affecting synaptic andcognitive function.

Therefore, what is needed are specific agonists that act upon thelipoprotein receptor system in a manner similar to Reelin for use astherapeutics in the improvement of cognitive function as well as thetreatment of neurological disease such as AD and other age-relatedneurodegenerative disorders.

SUMMARY OF INVENTION

The invention relates generally to methods of influencing, andenhancing, cognitive function by increasing, and/or preventinginterference with, Reelin levels as well as the cellular signaltransduction initiated or maintained with Reelin or Reelin signaling.

In a first embodiment, the invention includes a method of improvingcognitive function, in a subject in need thereof, by administering atherapeutically effective amount of Reelin, a Reelin-specific modulatoror an agonist of a lipoprotein receptor to the subject. The lipoproteinreceptor can be selected from candidates such as ApoER2 and VLDLR. Asdisclosed herein, agonists or antagonists of the lipoprotein receptorfor use with the inventive method include, but are not limited to, APC,Sep and Fc-RAP. In addition to administering exogenous Reelin, aReelin-specific modulator, such as a recombinant Reelin fragment, can beused to increase Reelin levels and/or signaling. In an illustrativeembodiment, the therapeutically effective amount of Reelin or an agonistof a lipoprotein receptor is approximately 5 nM.

In another embodiment, the invention includes a method of treating asymptom of a disease or disorder of the nervous system by administeringa therapeutically effective amount of Reelin, a Reelin-specificmodulator or an agonist of a lipoprotein receptor to a subject in needthereof. As with the previous embodiment, the lipoprotein receptor isselected from the group consisting of ApoER2 and VLDLR. The agonists ofthe lipoprotein receptor for use with the inventive method include, butare not limited to, APC, Sep and Fc-RAP. In this embodiment, the diseaseor disorder of the nervous system can be selected from the groupconsisting of fragile X syndrome, William's syndrome, Rett syndrome,Down's syndrome, Angelman syndrome, autism, ischemia, hypoxia,Alzheimer's disease, Reelin deficiency, schizophrenia,neurodegeneration, traumatic brain injury, mental retardation, dementia,and stroke. The therapeutically effective amount of Reelin or an agonistof a lipoprotein receptor is, in one example, approximately 5 nM.

A third embodiment of the invention includes a method of increasingdendritic spine density, in a subject in need thereof, by administeringa therapeutically effective amount of Reelin, a Reelin-specificmodulator or an agonist of a lipoprotein receptor to the subject. Thelipoprotein receptor can be selected from candidates such as ApoER2 andVLDLR. As disclosed herein, agonists of the lipoprotein receptor for usewith the inventive method include, but are not limited to, APC, Sep andFc-RAP. In addition to administering exogenous Reelin, a Reelin-specificmodulator, such as a recombinant Reelin fragment, can be used toincrease Reelin levels and/or signaling. In an illustrative embodiment,the therapeutically effective amount of Reelin or an agonist of alipoprotein receptor is about 5 nM.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1. Application of recombinant Reelin enhances LTP. Field recordingsfrom acute hippocampal slices in area CA1. Wild-type mice were perfusedwith either 5 nM reelin (n=7) or Mock (n=6).

FIG. 2A. Reelin enhances NMDAR currents through postsynaptic mechanisms.Illustration of measurement of EPSCNMDA.

FIG. 2B. Reelin enhances NMDAR currents through postsynaptic mechanisms.Illustration of measurement of EPSCNMDA. The thick gray trace representsthe mEPSCNMDA.

FIG. 2C. Reelin enhances NMDAR currents through postsynaptic mechanisms.Reelin treatment significantly increased mEPSCNMDA amplitude (closedcircle, before reelin; open circle, after reelin; ***p<0.001; n=18;paired t test). Treatment with mock was without effect [closed square,before mock; open square, after mock; not significant (ns), p>0.05;n=13; paired t test].

FIG. 2D. Reelin enhances NMDAR currents through postsynaptic mechanisms.No correlation of 1/CV2 ratios and mean EPSCNMDA ratios (after/beforereelin) was revealed based on recordings from nine cells (r=0.31; p=0.4;Spearman's test).

FIG. 3A. Reelin signaling alters surface expression and total levels ofglutamate receptor subunits. Representative blots showing levels of bothsurface and total GluR1, NR1, NR2A, and NR2B.

FIG. 3B. Reelin signaling alters surface expression and total levels ofglutamate receptor subunits.) Quantitative results of surface glutamatereceptor subunits pooled from 4 experiments. Compared with mock groups,both surface GluR1 and NR2A were significantly increased [GluR1,F(2,11)=15.56, ***P<0.001; NR2A, F(2,11)=44.9, ***P<0.001], and thelevel of surface NR2B was significantly reduced [F(2,11)=22.6,***P<0.001] after chronic Reelin treatment.

FIG. 3C. Reelin signaling alters surface expression and total levels ofglutamate receptor subunits. Reelin treatment significantly increasedlevels of total GluR1 [F(2,11)=11.2, **P<0.01], NR2A [F(2,14)=9.75,**P<0.01], and decreased level of total NR2B [F(2,11)=4.1, *P<0.05]. Incontrast, neither total nor surface (in 3B) levels of NR1 was observed.

FIG. 4A. Reelin supplementation can improve associative learning andspatial learning. Wild type mice were given either 5 nM RAP or 5 nMReelin by bilateral injection into the ventricles 3 hours prior toreceiving fear conditioning. 24 hrs after training, mice were placedinto the context and freezing measured. RAP was found to inhibitlearning and memory while Reelin led to an enhancement (RAP n=9, noshock n=5, no treatment n=7, Reelin n=5; p>0.05).

FIG. 4B. Reelin supplementation can improve associative learning andspatial learning. Wild type mice were trained to find a hidden platformthrough the Morris Water maze. Mice were given a single injection ofeither 5 nM Reelin (red circle, n=4) or Vehicle (open circle, n=6). Onday 5, a probe trial was given then the mice were trained to find a newplatform location on day 6.

FIG. 4C. Reelin supplementation can improve associative learning andspatial learning. Examination of latencies from individual trials onday 1. (*=p>0.05).

FIG. 4D. Reelin supplementation can improve associative learning andspatial learning. Wild type mice were trained to find a hidden platformthrough the Morris Water maze. Mice were given a single injection ofeither 5 nM Reelin (n=4) or Vehicle (n=6).

FIG. 5A. Reelin signaling is altered in AD mouse models. Isolatedcortices from 14-month old wild type, Tg2576 (SweAPP), PS1-FAD (M146L),and 2× (SweAPP×M146L) were subjected to western analysis (n=4). Nosignificant differences were detected in Reelin 450, 190 and 180 kDaproducts in Tg2576 versus wild type, but unidentified N-terminal speciesrecognized by G10 were significantly elevated in Tg2576 and 2× mice. Incontrast, Reelin 450 and 180 kDa products were significantly elevated inPS1-FAD and 2× mice (p<0.05).

FIG. 5B. Reelin signaling is altered in AD mouse models. There weresignificant reductions in Dab1-pTyr220 in Tg2576 mice, and significantelevations in both PS1-FAD and 2× mice.

FIG. 5C. Reelin signaling is altered in AD mouse models. Application ofReelin (5 nM) prior to stimulation was able to rescue deficits inHFS-stimulated LTP in area CA1 of Tg2576 mice.

FIG. 5D. Reelin signaling is altered in AD mouse models. The 3-epitopestrategy for mapping Reelin processing in vivo was employed on 14-monthold Tg2576 horizontal sections. Reelin-CT (G20). Scale bar=15 μm.

FIG. 5E. Reelin signaling is altered in AD mouse models. The 3-epitopestrategy for mapping Reelin processing in vivo was employed on 14-monthold Tg2576 horizontal sections. Reelin-NT. Scale bar=15 μm.

FIG. 5F. Reelin signaling is altered in AD mouse models. The 3-epitopestrategy for mapping Reelin processing in vivo was employed on 14-monthold Tg2576 horizontal sections. Reeling MT (AF3820), detected Reelinfragments containing R7-8 and R3-6, respectively, sequestered at thecore of a dense-core plaque detected with 6E10 (anti-Aβ). Scale bar=15μm.

FIG. 5G. Reelin signaling is altered in AD mouse models. The 3-epitopestrategy for mapping Reelin processing in vivo was employed on 14-monthold Tg2576 horizontal sections. Reelin-CT (G20), -NT, -MT (AF3820) weremerged, detecting Reelin fragments containing R7-8 and R3-6,respectively, sequestered at the core of a dense-core plaque detectedwith 6E10 (anti-Aβ). Scale bar=15 μm.

FIG. 5H. Reelin signaling is altered in AD mouse models. The 3-epitopestrategy for mapping Reelin processing in vivo was employed on 14-monthold Tg2576 horizontal sections. Reelin-NT fragments (N-R2) surroundedthe plaque core in the tg2576 mouse model. Scale bar=15 μm.

FIG. 6. LTP induction using a standard 2-train, 100 Hz HFS was given tohippocampal slices from 12 month-old Tg2576 mice. A set of slices wereperfused with 5 nM reelin. Reelin treated slices showed an increase ofLTP induction to that of wild-type levels.

FIG. 7. Targeted deletion of the Selenoprotein P gene results in LTPdeficit. Field recordings of acute hippocampal slices show no LTP after100 Hz stimulation is given (blue arrow) SeP (−/−) n=12, SeP (+/+) n=8.Peters et at 2006

FIG. 8. Addition of Selenoprotein P rescues the LTP deficit in micelacking the Selenoprotein P gene. Field recordings of acute hippocampalslices in SeP (−/−). Slices treated with 2 nM SeP for 20 min (red line)then given 100 Hz stimulation. SeP (−/−)+2 nM SeP n=16, SeP (−/−) no SePn=28.

FIG. 9. Perfusion with Fc-RAP enhances hippocampal LTP induction.Hippocampal slices were perfused with Fc-RAP (10 μg/ml), Fc (10 μg/ml),or control medium. Baseline synaptic responses (marked by *) andpotentiation immediately following HFS (marked by †) and up to 60 minafter HFS (marked by ♦) were recorded. The arrowhead represents LTPinduced with two trains of 1-s-long, 100-Hz stimulation, separated by 20s. The horizontal line indicates application of Fc-RAP, Fc, or controlmedium. Results are shown as means±standard errors of the mean. fEPSP,field excitatory postsynaptic potential Strasser et al 2004.

FIG. 10A. Contextual fear conditioning alters Reelin levels. Wild typemice were trained with a 3-shock, contextual fear conditioning protocol(CFC). Non-shocked mice (CS) were used as a negative control andshocked, context-exposed mice (CS/US) had their hippocampus removed at1, 5, 15, 30, and 180 minutes after training, as well as 18 hourspost-training (n=4, time point). Reelin was detected in hippocampalhomogenates using anti-Reelin (G10).

FIG. 10B. Contextual fear conditioning alters Reelin levels. Wild typemice were trained with a 3-shock, contextual fear conditioning protocol(CFC). Non-shocked mice (CS) were used as a negative control andshocked, context-exposed mice (CS/US) had their hippocampus removed at1, 5, 15, 30, and 180 minutes after training, as well as 18 hourspost-training (n=4, time point). Reelin was detected in hippocampalhomogenates using anti-Reelin (G10) and the levels of full-length Reelinwere quantitated. The asterisks denote statistical significancefollowing a two-tailed t-test, where p<0.5.

FIG. 11A. HFS alters Reelin metabolism in a tPA-dependent manner. Acutehippocampal slices were stimulated using TB-STIM (theta burststimulation) consisting of 5 trains at theta-burst across the Schaffercollateral. Hippocampi were harvested 15 minutes later and homogenateswere subjected to western blot analysis and detected with anti-Reelin(G10) (n=3 per group). Non-stimulated is denoted as NS and stimulated asS.

FIG. 11B. HFS alters Reelin metabolism in a tPA-dependent manner. Agraph showing quantified results of acute hippocampal slices stimulatedusing TB-STIM (theta burst stimulation) consisting of 5 trains attheta-burst across the Schaffer collateral. Hippocampi were harvested 15minutes later and homogenates were subjected to western blot analysisand detected with anti-Reelin (G10) (n=3 per group). Non-stimulated isdenoted as NS and stimulated as S. The 370 kDa was quantified andstatistically analyzed using a two tailed t-test (*, p<0.05).

FIG. 12A. tPA modulates Reelin processing. The ability oftPA/plasminogen to affect Reelin processing was determined by reactingReelin (50 nM) with tPA (60 ug/ml), inactive plasminogen (18 ug/ml), tPAand plasminogen, and Plasmin (active, 0.5 U/ml) in PBS for 45 minutes at37° C. Reactions were run on Westerns (at 1:10) and probe withanti-Reelin (G10, an N-R2 recognizing antibody) and ant-I Reelin (Ab14,a R7-8 recognizing antibody).

FIG. 12B. tPA modulates Reelin processing. The ability of tPA to affectReelin metabolism in primary cortical neurons was determined byincubating cells in fresh supernatant for 24 hours with 70 nM tPA forcellular extracts subjected to Western analysis and detection with G10.

FIG. 12C. tPA modulates Reelin processing. The ability of tPA to affectReelin metabolism in primary cortical neurons was determined byincubating cells in fresh supernatant for 24 hours with 70 nM tPA forsupernatant protein extracts subjected to Western analysis and detectionwith G10.

FIG. 12D. MMP-9 modulates Reelin processing. The ability of MMP-9(active; Calbiochem, PF140) to affect Reelin processing was determinedby reacting Reelin (50 nM) with different concentrations of MMP-9 (1-4ug/ml) in PBS at 37° C. for 3 hours. EDTA (10 mM) was included as anegative control, as it blocks MMP9 activity. Western blots were run on1:10 of the reaction and probed with anti-Reelin (G10). The ability ofMMP-9 (250 nM) and the MMP-9 inhibitor (25 nM; Calbiochem 444278) toaffect Reelin processing in primary cortical neurons was determinedafter 24 hours in both cellular and supernatant extracted proteins.

FIG. 12E. MMP-9 modulates Reelin processing. The ability of MMP-9(active; Calbiochem, PF140) to affect Reelin processing was determinedusing the cellular fraction by reacting Reelin (50 nM) with differentconcentrations of MMP-9 (1-4 ug/ml) in PBS at 37° C. for 3 hours.Western blots were run on 1:10 of the reaction and probed withanti-Reelin (G10). The ability of MMP-9 (250 nM) and the MMP-9 inhibitor(25 nM; Calbiochem 444278) to affect Reelin processing in primarycortical neurons was determined after 24 hours in the cellular fraction.

FIG. 12F. MMP-9 modulates Reelin processing. The ability of MMP-9(active; Calbiochem, PF140) to affect Reelin processing was determinedusing the supernatant protein fraction by reacting Reelin (50 nM) withdifferent concentrations of MMP-9 (1-4 ug/ml) in PBS at 37° C. for 3hours. Western blots were run on 1:10 of the reaction and probed withanti-Reelin (G10). The ability of MMP-9 (250 nM) and the MMP-9 inhibitor(25 nM; Calbiochem 444278) to affect Reelin processing in primarycortical neurons was determined after 24 hours in the supernatantextracted proteins.

FIG. 12G. MMP-9 modulates Reelin processing. The ability of MMP-9(active; Calbiochem, PF140) to affect Reelin processing was determinedusing the cellular fraction by reacting Reelin (50 nM) with differentconcentrations of MMP-9 (1-4 ug/ml) in PBS at 37° C. for 3 hours alongwith EDTA (10 mM) as a negative control, as it blocks MMP9 activity.Western blots were run on 1:10 of the reaction and probed withanti-Reelin (G10). The ability of MMP-9 (250 nM) and the MMP-9 inhibitor(25 nM; Calbiochem 444278) to affect Reelin processing in primarycortical neurons was determined after 24 hours in the cellular fraction.

FIG. 12H. MMP-9 modulates Reelin processing. The ability of MMP-9(active; Calbiochem, PF140) to affect Reelin processing was determinedusing the supernatant protein fraction by reacting Reelin (50 nM) withdifferent concentrations of MMP-9 (1-4 ug/ml) in PBS at 37° C. for 3hours along with EDTA (10 mM) as a negative control, as it blocks MMP9activity. Western blots were run on 1:10 of the reaction and probed withanti-Reelin (G10). The ability of MMP-9 (250 nM) and the MMP-9 inhibitor(25 nM; Calbiochem 444278) to affect Reelin processing in primarycortical neurons was determined after 24 hours in the supernatantextracted proteins.

FIG. 13A. Tri-epitope mapping. Reelin consists of an N-terminal regionfollowed by the CR-50 electrostatic domain (purple), an F-spondin domain(H), and 8 consecutive EGF-like repeats.

FIG. 13B. Tri-epitope mapping. Antibodies that distinctly recognize theN-R2, R3-R6, and R7-R8 regions of Reelin can be used to determine thedistribution of full-length Reelin and its major fragments.

FIG. 13C. Tri-epitope mapping. Antibodies that will be employed in the3-epitope approach are listed.

FIG. 14. Illustration of constructs to be used in SA2 and SA3 and sitesof Reelin cleavage. MMP-9 can cleave between regions 2 and 3, but hasalso been shown to cleave in region 7 during in vitro reactions only.tPA can cleave between regions 6 and 7. Proposed constructs are madewithout the in vitro MMP-9 binding site a with both C and N terminaltags. Rln-Res=Reelin Cleavage Resistant; Rln-Lab=Reelin labile.

FIG. 15A. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines on a WT neuron areshown in an enlarged photo of a representative primary dendrite. Thelast column of reelin represents the native in the concentrationadministered to the culture. Reelin was present up until 96 hours afterintroduction to culture and degradation did not begin until 72 hours.

FIG. 15B. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines are reduced in theHRM compared to WT mice but after treatment with reelin, spine densityis rescued. The last column of reelin represents the native in theconcentration administered to the culture. Reelin was present up until96 hours after introduction to culture and degradation did not beginuntil 72 hours.

FIG. 15C. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines are reduced in theHRM compared to WT mice but after treatment with reelin, spine densityis rescued. The last column of reelin represents the native in theconcentration administered to the culture. Reelin was present up until96 hours after introduction to culture and degradation did not beginuntil 72 hours.

FIG. 15D. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines are very sparse inthe knockout reelin mice but after treatment with reelin, spine densitydeficits are rescued. The last column of reelin represents the native inthe concentration administered to the culture. Reelin was present upuntil 96 hours after introduction to culture and degradation did notbegin until 72 hours.

FIG. 15E. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines are very sparse inthe knockout reelin mice but after treatment with reelin, spine densitydeficits are rescued. The last column of reelin represents the native inthe concentration administered to the culture. Reelin was present upuntil 96 hours after introduction to culture and degradation did notbegin until 72 hours.

FIG. 15F. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Dendritic spines were quantifiedusing a confocal microscope. Dendritic spines were defined as anyprotrusion from a primary dendrite excluding any secondary dendrites.Dendritic spines were counted and measured every 50 um of the dendrite.There is a significant increase in spines in reelin-treated cells (n=3)versus mock-treated cells (n=3). The last column of reelin representsthe native in the concentration administered to the culture. Reelin waspresent up until 96 hours after introduction to culture and degradationdid not begin until 72 hours.

FIG. 15G. Reelin effects on dendritic spine density. Reelin was appliedchronically to primary hippocampal neuronal cultures to examine itseffect on dendritic spine density. Reelin levels in culture weredetermined by a Western Blot. Samples were taken out of culture at 0, 6,12, 24, 48, 72, and 96 hrs to determine the levels of reelin degradationin vitro. The last column of reelin represents the native in theconcentration administered to the culture. Reelin was present up until96 hours after introduction to culture and degradation did not beginuntil 72 hours.

FIG. 16A. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Open field behavior utilized to evaluate locomotoractivity. The total distance traveled during the 15 min test was similarfor the three conditions (mock HRM n=13, reelin HRM n=13, Rap WT n=10;ANOVA p=0.23).

FIG. 16B. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Elevated Plus Maze utilized to determine anxiety. Thepercent of time spent in the open arms were similar for the threeconditions (mock HRM n=11, reelin HRM n=10, Rap WT n=13; percent timeANOVA p=0.49 and open arm entries ANOVA p=0.63).

FIG. 16C. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Elevated Plus Maze utilized to determine anxiety. Thenumber of open arm entries were similar for the three conditions (mockHRM n=11, reelin HRM n=10, Rap WT n=13; percent time ANOVA p=0.49 andopen arm entries ANOVA p=0.63).

FIG. 16D. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Prepulse inhibition utilized to evaluate startleresponse. The startle response to a 120 dB acoustic stimulation issimilar for the three conditions (mock HRM n=14, reelin HRM n=16, Rap WTn=15; ANOVA p=0.56). Results from Qiu et al. (2005) are depicted withdashed lines for reference.

FIG. 16E. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Acoustic startle utilized to evaluate startle response.The startle response to a 120 dB acoustic stimulation is similar for thethree conditions (mock HRM n=14, reelin HRM n=16, Rap WT n=15; ANOVAp=0.56). Results from Qiu et al. (2005) are depicted with dashed linesfor reference.

FIG. 17A. HRM contextual fear conditioning deficits are rescued byapplication of exogenous reelin. Freezing during the conditioningparadigm was similar for both conditions (mock HRM n=16, reelin HRMn=16). The tone is represented by the black bar and the shock by theblack arrows. Freezing during reintroduction to the conditioningcontext.

FIG. 17B. HRM contextual fear conditioning deficits are rescued byapplication of exogenous reelin. Freezing was similar for the threeconditions 1 hr post conditioning (mock HRM n=13, reelin HRM n=13).

FIG. 17C. HRM contextual fear conditioning deficits are rescued byapplication of exogenous reelin. Reelin-treated HRM freezing wassignificantly greater than mock-treated HRM 24 hrs post conditioning(mock HRM n=16, reelin HRM n=16; t-test p=0.02).

FIG. 17D. HRM contextual fear conditioning deficits are rescued byapplication of exogenous reelin. Reelin-treated HRM freezing wassignificantly greater than mock-treated HRM 72 hrs post conditioning(mock HRM n=5, reelin HRM n=4; t-test p=0.026).

FIG. 17E. HRM contextual fear conditioning deficits are rescued byapplication of exogenous reelin. Shock threshold analysis to evaluatenociception. The shock intensity in which mice flinched, jumped, orvocalized was similar for both conditions (mock HRM n=3, reelin HRM n=3;ANOVA p=0.22).

FIG. 18A. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Open field behavior utilized to evaluate locomotoractivity. The total distance traveled during the 15 min test was similarfor the two conditions (Rap WT n=10).

FIG. 18B. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Elevated Plus Maze utilized to determine anxiety. Thepercent of time spent in the open arms were similar for both conditions(Rap WT n=13; percent time ANOVA p=0.49 and open arm entries ANOVAp=0.63).

FIG. 18C. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Elevated Plus Maze utilized to determine anxiety. Thenumber of open arm entries were similar for both conditions (Rap WTn=13; percent time ANOVA p=0.49 and open arm entries ANOVA p=0.63).

FIG. 18D. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Prepulse inhibition, Results from Qiu et al. (2005) aredepicted with dashed lines for reference.

FIG. 18E. Locomotor activity, nociception and anxiety are unaltered bydrug treatments. Acoustic startle utilized to evaluate startle response.The startle response to a 120 dB acoustic stimulation is similar forboth conditions (Rap WT n=15; ANOVA p=0.56).

FIG. 19A. Freezing during the conditioning paradigm was similar for bothconditions (Rap WT n=13). The tone is represented by the black bar andthe shock by the black arrows. Freezing during reintroduction to theconditioning context.

FIG. 19B. Freezing was similar for both conditions 1 hr postconditioning (Rap WT n=9).

FIG. 19C. RAP-treated WT freezing was significantly less thanvehicle-treated WT (Rap WT n=13) 24 hrs post conditioning There was nodifference between mock-treated HRM freezing and Rap-treated WT freezingat any time tested (See FIGS. 15B-C).

FIG. 19D. Freezing was similar for both conditions 1 hr postconditioning (Rap WT n=9). (C) RAP-treated WT freezing was significantlyless than vehicle-treated WT (Rap WT n=13) 72 hrs post (Rap WT n=6).There was no difference between mock-treated HRM freezing andRap-treated WT freezing at any time tested (See FIGS. 15B-C).

FIG. 19E. Shock threshold analysis to evaluate nociception. The shockintensity in which mice flinched, jumped, or vocalized was similar forboth conditions (Rap WT n=4; ANOVA p=0.22).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Recent research has established a role for lipoprotein receptors incognitive processes and implicated this receptor family in thepathological processes that underlie the progression of AD. Two of themajor ligands for these receptors, apoE and reelin, appear to havesignaling capabilities that can significantly impact synaptic function,directly interact with APP and modulate its metabolism, and aresensitive to Aβ accumulation. Aβ accumulation disrupts lipoproteinreceptor signaling, resulting in concomitant disruption of cognitivefunction. Furthermore, interference of reelin and/or lipoproteinreceptor signaling results in aberrant APP metabolism and Aβ clearancethat in turn exacerbates Aβ accumulation and plaque deposition.Therefore, increased reelin signaling through direct reelin applicationor usage of other lipoprotein receptor agonists can be used to mitigateAβ-dependent cognitive disruption and progression of plaque pathology.

Reelin: In the adult hippocampus, the glycoprotein Reelin is expressedby interneurons residing primarily in the hilar region of dentate gyrus,and the stratum lacunosum-moleculare layer of the hippocampus proper.Reelin-expressing cells can also be found in stratum oriens and stratumradiatum of area CA1 and CA3 and is associated with pyramidal cells ofthe hippocampus. Induction of long-term potentiation (LTP), a form ofsynaptic plasticity that results in a lasting increase in synapticefficacy, requires NMDAR (NMDARs) activation and the subsequentup-regulation of AMPA receptor expression and function. Changes in AMPAreceptors (AMPARs) can be achieved either by increased subunitphosphorylation or by increased subunit synthesis and trafficking to thespecific synaptic sites. In contrast, NMDARs serve as coincidencedetectors and play a major role in the induction of synaptic plasticity.The opening of NMDAR ion channels requires both glutamate binding andpost-synaptic membrane depolarization. Some NMDAR subunits, such as NR1,NR2A and NR2B are also subjected to modulatory phosphorylation atserine/threonine or tyrosine residues. Phosphorylation of NMDAR subunitsmodulates both channel kinetics and trafficking to synaptic sites. Itfollows that if reelin were important for modulation of synapticplasticity, then NMDARs and AMPARs would be logical targets given theirimportance in induction and expression of synaptic plasticity.

APC: Activated protein C (APC) is a serine protease that possesses bothanticoagulant and cytoprotective properties that are currently beingexploited for the treatment of conditions such as sepsis, stroke andmultiple sclerosis. The anticoagulant properties of APC are achievedthrough the protein C (PC) pathway, while its cryoprotective effects areorchestrated through PAR1 (protease activated receptor; and PAR3,endothelial PC receptor (EPCR) and ApoER2. In mice, APC has been foundto protect against diabetic endothelial and glomerular injury, multiplesclerosis and ischemia/reperfusion injury in the kidney and lung.

APC has already been approved by the U.S. Food and Drug Administrationfor use in adult severe sepsis and is currently in Phase I/IIa clinicaltrials for the treatment of ischemic stroke (National Institutes ofHealth, Activated Protein C in Acute Stroke Trial (APCAST), 2010).Numerous groups have also recently developed APC variants that possessless anticoagulant activity, which has proven to limit APC's clinicalefficacy. Specifically, a mutant designated 3K3A-APC has 80% reducedanticoagulant activity but retains normal PAR1 and EPCR-dependentanti-apoptotic activity. Relevant to the use of APC to treatneuropathologies, APC and APC variants have been found to effectivelycross the BBB via EPC-mediated transport.

Recently, APC has been found to activate the Reelin signaling cascadevia high affinity ligation to ApoER2. Specifically, APC-treatedmonocytes demonstrated increased active Dab1 (Tyr220-p), Akt Ser473-p,and GSK3beta Ser9-p levels. Pre-treatment with RAP or knocking down ofApoER2 were found to attenuate these effects, while inhibitors of EPCRand PAR1 had no effect. Interestingly, APC was found to bind to ApoER2with 30 nM affinity, but not to soluble VLDLR. To relate APC's effectsto ApoER2 signaling, RAP was found to block APC-mediated inhibition ofendotoxin-induced tissue factor pro-coagulant activity of U937 cells.

Recent work has highlighted the importance of Reelin signaling in normallearning and memory (Weeber E J, Beffert U, Jones C, et al. Reelin andApoE receptors cooperate to enhance hippocampal synaptic plasticity andlearning. J Biol Chem 2002, 277:39944-39952), as well as pathologicalinstances where this signaling is perturbed. APC is now a candidatemodulator of Reelin signaling, as it appears to have the structuralmoieties to bind to ApoER2 and activate downstream effectors. It is ofimmense scientific and clinical relevance that APC modulation of Reelinsignaling be tested, as it could yield novel therapeutic avenues.

SePP1: Approximately 60% of selenium in plasma is present inselenoprotein P. This protein differs from other selenoproteins in thatit incorporates up to 10 Se atoms per molecule in the form ofselenocysteine as opposed to single selenocysteines. Selenoprotein P isabundant throughout the body, suggesting that one function is to serveas a primary transporter in systemic selenium delivery. This isespecially evident in the CNS where selenoprotein P levels can bemaintained independent of plasma selenium. However, genetic ablation ofselenoprotein P results in reduced, but not a commensurate decrease inCNS-associated selenium levels, suggesting that other selenium proteinscompensate for the selenoprotein P deficiency and supporting thehypothesis that basal selenium levels are essential for the brain andhave a priority for systemically available selenium. Sepp1 (−/−) micefed a selenium-deficient diet show severe motor dysfunction associatedand associated neuronal degeneration, which can be prevented bysupplementation with high dietary selenium.

Reduced dietary selenium can have significant effects on levels ofselenoproteins involved in oxidative stress and their related effects onglutathione peroxidases, thioredoxin reductases and methionine sulfoxidereductases. Selenium, through incorporation into selenoproteins,provides protection from reactive oxygen species (ROS)-induced celldamage. This is interesting in light of the role of oxidative stress andsubsequent production of ROS in neurodegenerative disorders such asAlzheimer's disease, Parkinson's disease and Duchenne musculardystrophy. The inventors have previously examined the consequences ofselenoprotein P deficiency on cognitive capacity and synaptic functionwith a focus on the hippocampus, an area of the CNS intimately involvedin learning and memory processes. Sepp1 (−/−) mice demonstrated no overtbehavioral phenotype, but were found to have a subtle disruption inacquisition of spatial learning and memory. In contrast, synaptictransmission was altered and short- and long-term synaptic plasticitywas severely disrupted in area CA1 of hippocampus. Interestingly, theinventors found that when Sepp1 (+/+) mice were fed a low Selenium diet(0 mg/kg), they too exhibited altered synaptic transmission and synapticplasticity. Our observations suggest an important role for bothselenoprotein P and dietary selenium in overall proper synapticfunction.

Fc-RAP: Reelin molecules have recently been discovered to formhigher-order complexes in vitro and in vivo. This observation wasfurther refined by showing that reelin is secreted in vivo as adisulfide-linked homodimer. Deletion of a short region, called the CR-50epitope, located at the N-terminus of the molecule abolishesoligomerization. This mutated reelin fails to efficiently induce Dab1phosphorylation in primary mouse neurons.

These results are in accordance with earlier observations that anantibody against the CR-50 epitope antagonizes reelin function in vitroand in vivo. Clustering of ApoER2 and/or VLDLR induces Dab1phosphorylation and downstream events including activation of SFKs andmodulation of PKB/Akt. Furthermore, modulation of long-term potentiation(LTP), one of the biological effects of reelin, is also mimicked byreelin-independent receptor clustering. These findings strongly suggestthat receptor-induced dimerization or oligomerization is sufficient forDab1 tyrosine phosphorylation and downstream signaling events withoutthe need for an additional co-receptor providing tyrosine kinaseactivity.

As shown herein, Reelin plays an active role in the processes ofsynaptic plasticity and learning. The invention also includes theidentification and use of mechanisms for Reelin protein processing toenhance and/or repair cognitive function. For example, it is disclosedherein that: contextual fear learning and theta burst stimulation(tb-stim) cause changes in Reelin processing; the metalloproteinases,tPA and MMP-9 are differentially involved in Reelin processing duringsynaptic plasticity and learning; supplementation of Reelin fragmentcomplement can enhance associative and spatial learning and memory; andreelin fragments associate with Aβ plaques, its expression andprocessing is altered by AD-related mutations, and Reelinsupplementation can overcome the LTP deficits found in the Tg2576 ADmouse model.

Reelin-Induced Enhancement of Long-Term Potentiation in AcuteHippocampal Slices.

Reelin is a naturally occurring, secreted protein produced byinterneurons of the hippocampus and cortex. Knockout (KO) mice of bothreelin receptors, ApoER2 and VLDLR show deficits in long-termpotentiation (LTP) in the stratum radiatum of the hippocampus. To verifythe absence of reelin signaling underlies this deficit, the inventorsperformed a simple experiment consisting of the perfusion of purifiedreelin protein onto wild-type hippocampal slices. As shown in FIG. 1,reelin application enhanced HFS-LTP induced in the stratum radiatum.

Post-Synaptic Mechanisms of Reelin Enhancement of NMDAR Currents.

Reelin also demonstrates the ability to potentiate CA1 glutamatergicresponses. The inventors have recently shown that ApoER2 is presentpost-synaptically and forms a functional complex with NMDARs in CA1 (4).The derivation of mEPSCNMDA is illustrated in FIG. 2. Cells treated withmock had miniature excitatory post-synaptic current due to NMDAreceptors (mEPSCNMDA) that were not significantly changed compared withthat before mock treatment (p>0.05). Treatment with Reelin was found tosignificantly increase mEPSCNMDA amplitude (p<0.001).

To further verify that synaptic NMDAR response was increased as a resultof postsynaptic effects of Reelin, the inventors analyzed thecoefficient of variation (CV) of synaptically-evoked NMDAR whole-cellcurrent. When 1/CV2 ratios were plotted versus mean EPSCNMDA ratiosbefore and after a 30 minute reelin application in nine experiments, nocorrelation was established (FIG. 2D). However, the 1/CV2 ratios remainrelatively unchanged across varying mean EPSCNMDA ratios, confirmingreelin activation through a postsynaptic mechanism in CA1 to enhanceNMDAR activity.

Differential Effects of Reelin Treatment on Surface Levels of AMPAR andNMDAR subunits.

Chronic Reelin treatment can result in the increased AMPA component ofsynaptic response, alteration of EPSCNMDA kinetics and ifenprodilsensitivity. The inventors sought to determine whether the proteinexpression levels of AMPAR and NMDAR subunits were changed by Reelin inCA1. Both total and surface levels of GluR1, NR1, NR2A, and NR2B wereprobed by Western blotting. The inventors first examined whether GluR1,an AMPAR subunit that is increasingly expressed during developmentalmaturation and subjected to regulate trafficking during synapticplasticity, was increased on CA1 cell surfaces.

FIG. 3 shows that reelin treatment significantly increased levels ofsurface GluR1 compared with mock-treated groups, indicating regulatedexpression and surface insertion via increased mEPSC_(AMPA) andAMPA/NMDA current ratio after chronic Reelin treatment. No changes ofeither surface or total NR1 levels were observed. In comparison, bothtotal and surface NR2A expression levels were significantly increasedafter reelin treatment versus mock treatment. Moreover, both total andsurface NR2B protein levels were significantly decreased followingreelin treatment. Mock treatment had no effect on different glutamatereceptor subunit levels compared with non-treated control groups.

Reelin signaling translates from a role in synaptic plasticity tolearning and memory.

Reelin heterozygotes show deficits in both synaptic plasticity andcognitive function. An approximate 50% reduction of Reelin expressionresults in deficits in both synaptic plasticity and cognitive function(Qiu, S., K. M. Korwek, A. R. Pratt-Davis, M. Peters, M. Y. Bergman, andE. J. Weeber. 2006. Cognitive disruption and altered hippocampussynaptic function in Reelin haploinsufficient mice. Neurobiol Learn Mem85:228-242). Furthermore, bilateral infusion of the lipoproteinantagonist RAP (receptor associated protein), which effectively blocksReelin binding to its receptors dramatically, reduced associativelearning (FIGS. 4A-4D). These results demonstrate a requirement forReelin for normal memory formation and raise the interesting question ofwhether increasing Reelin signaling can enhance memory.

The effect of Reelin deficiency on synaptic function is contrasted whenReelin concentrations are enhanced. Direct bilateral ventricle infusionof recombinant Reelin fragment compliment 3 hours prior to associativefear conditioning training enhanced memory formation when tested 24hours after training in 3-4 month-old wild-type mice (FIG. 4A).

Furthermore, a single injection of Reelin into the ventricles improvedspatial learning in the hidden platform water maze (FIG. 4B). Mice thatwere retrained to find a different platform location (opposite) on day 6continued to show increased learning ability compared to saline injectedmice. Mice receiving a single Reelin injection 5 days prior to trainingshow a lower latency to find the platform on day one. A closerexamination shows that the latency to find the platform is significantlyreduced after a single exposure to the training paradigm (FIG. 4C). Micethat were retrained to find a different platform location continued toshow differences between reelin and saline injections. Swim speeds andall other measurements of activity between treated and non-treatedanimals remained the same. This data dramatically illustrate the abilityof Reelin to modulate in vivo learning and memory formation and theimportance of research aimed to identify the mechanisms controllingReelin protein processing and how the fragments subsequently modulatecognitive function.

Reelin Supplementation Overcame Aβ-Dependent Changes in SynapticPlasticity.

Reelin signaling is involved in a variety of physiologic changes to theexcitatory synapse, as well as normal mammalian cognitive function.Reelin metabolism is altered in three mouse models for AD (PS1-FAD,SweAPPxPS1, and Tg2576) (FIG. 5A). These changes in Reelin fragmentcomplement appear to be correlated with alterations in downstream Reelinsignaling, as phosphorylation of the major downstream component, Dab-1,is increased in the SweAPPXPS1 and PS1-FAD, and significantly decreasedin the single SweAPP (Tg2576) mouse (FIG. 5B). These data suggest thatReelin metabolism is particularly sensitive to changes in APP processingand/or Aβ accumulation.

The alteration in Reelin fragment complement and Dab-1 phosphorylationin the Tg2576 mice may represent a compromised Reelin signaling system,a phenomenon that if true could be responsible for the synapticplasticity deficits reported in these mice (Mitchell, J. C., B. B.Ariff, D. M. Yates, K. F. Lau, M. S. Perkinton, B. Rogelj, J. D.Stephenson, C. C. Miller, and D. M. McLoughlin. 2009. X11beta rescuesmemory and long-term potentiation deficits in Alzheimer's disease APPsweTg2576 mice. Hum Mol Genet 18:4492-4500; Kotilinek, L. A., M. A.Westerman, Q. Wang, K. Panizzon, G. P. Lim, A. Simonyi, S. Lesne, A.Falinska, L. H. Younkin, S. G. Younkin, M. Rowan, J. Cleary, R. A.Wallis, G. Y. Sun, G. Cole, S. Frautschy, R. Anwyl, and K. H. Ashe.2008. Cyclooxygenase-2 inhibition improves amyloid-betamediatedsuppression of memory and synaptic plasticity. Brain 131:651-664;Jacobsen, J. S., C. C. Wu, J. M. Redwine, T. A. Comery, R. Arias, M.Bowlby, R. Martone, J. H. Morrison, M. N. Pangalos, P. H. Reinhart, andF. E. Bloom. 2006. Early-onset behavioral and synaptic deficits in amouse model of Alzheimer's disease. Proc Natl Acad Sci USA103:5161-5166). Acute hippocampal slices from 8 month-old Tg2576 micewere perfused with 5 nM recombinant Reelin fragment complement. Theinventors find that the Reelin application rescues the LTP defect inaged Tg2576 mice (FIG. 5C) suggesting that the biochemical andstructural machinery involved in Reelin signaling downstream of Reelinprotein processing is intact in these mice. Furthermore, it is importantto note that normal levels of synaptic plasticity are obtainable in thismouse model. Reelin fragments are also associated with dense coreplaques in aged (15 month-old) Tg2576 mice (FIG. 5D-G). As shown in FIG.6, reelin and related lipoprotein receptor agonists can rescue deficitsin synaptic plasticity and cognitive function that result from Aβaccumulation and/or plaque pathology. Reelin rescued the LTP deficit in12 month-old mice modeled for AD (Tg2576) (FIG. 6).

These data are supported by Reelin associated with Aβ-containing plaquesdetected in the hippocampus of aged wild-type mice (Madhusudan, A., C.Sidler, and I. Knuesel. 2009. Accumulation of reelin-positive plaques isaccompanied by a decline in basal forebrain projection neurons duringnormal aging. Eur J Neurosci 30:1064-1076; Knuesel, I., M. Nyffeler, C.Mormede, M. Muhia, U. Meyer, S. Pietropaolo, B. K. Yee, C. R. Pryce, F.M. LaFerla, A. Marighetto, and J. Feldon. 2009. Age-related accumulationof Reelin in amyloidlike deposits. Neurobiol Aging 30:697-716). In lightof the established role for Reelin in synaptic function, changes in theintegrity of Reelin metabolism and signaling plays a profound role inthe learning and memory changes previously established in AD mousemodels.

Other ligands of lipoprotein receptors have an effect on synapticfunction.

Selenium containing Selenoprotein P (SeP) has been identified as anotherligand for the lipoprotein receptor ApoER2. SEP has been shown toassociate with ApoER2 in the testis and in the CNS. SeP KO mice showedvarious pathologies, including deficits in hippocampal-dependent LTP andcognitive function (FIG. 7).

Interestingly, the LTP defect in SeP (−/−) mice can be rescued withpurified SeP protein supplementation (FIG. 8). Taken together, thesedata suggest that SeP has a similar role to Reelin by signaling throughApoER2. It is unclear whether SeP can promote receptor clustering orcompete with Reelin. However, it appears that SeP is using the ApoER2 asa receptor to internalize the SeP and deliver selenium to the neuron.

Receptor Associated Protein (RAP) is an intracellular protein that canbind with very high affinity to the family of lipoprotein receptors. TheFc-RAP fusion protein is an engineered protein consisting of two RAPmolecules connected to form a rough ‘dumb bell’ shape using the Fcregion of an antibody. Instead of binding to and inhibiting ApoER2 andVLDLR, the Fc-RAP can cause receptor clustering and ApoER2 activation.The addition of Fc-RAP has the identical effect as reelin application byincreasing LTP induction (FIG. 9). The main difference is that theFc-RAP is likely to bind all lipoprotein receptors, but only clustersApoER2 and VLDLR.

Reelin fragment complement in the hippocampus is altered following invivo memory formation and ex-vivo stimulation.

Reelin is cleaved at specific sites resulting in a stable pattern ofReelin fragments easily quantified by Western blot analysis. Thesefragments represent potential signaling molecules with properties uniquefrom full-length Reelin. Recombinant Reelin purified from stablytransfected HEK293 cells contains fragments of the same size as themajor fragments found in the hippocampus. Application of recombinantReelin fragment compliment can (1) increase synaptic transmission byfacilitating AMPA receptor insertion and increasing NMDA receptorfunction, (2) reduce silent synapses, (3) modify synaptic morphology and(4) enhance LTP (Qiu, S., and E. J. Weeber. 2007. Reelin signalingfacilitates maturation of CA1 glutamatergic synapses. J Neurophysiol97:2312-2321; Qiu, S., K. M. Korwek, A. R. Pratt-Davis, M. Peters, M. Y.Bergman, and E. J. Weeber. 2006. Cognitive disruption and alteredhippocampus synaptic function in Reelin haploinsufficient mice.Neurobiol Learn Mem 85:228-242).

Additionally, fear conditioned learning produces changes in theendogenous Reelin fragment complement. The inventors found a dramaticchange in Reelin expression and fragment complement over the 18 hoursfollowing contextual fear conditioning, particularly in the 450 and 180kDa fragments (FIG. 10).

Moreover, theta burst stimulation delivered to the Schaffer collateralpathway led to significant increases in Reelin expression and fragmentcleavage at 15 minutes post-stimulation (FIG. 10). These results showthat integration and control of Reelin signaling responsible foralterations in synaptic plasticity and modulation of learning and memoryinvolves the processing of Reelin into functionally-distinct fragments.

The inventors also found that the efficacy of generating the 370 kDaproduct to be partially dependent on a candidate Reelin-cleaving enzyme,tPA. This potential mechanism of regulation has profound implications onhow this signaling system is integrated into known mechanisms ofneuronal regulation and coordinated to participate in physiologicalprocesses such as learning and memory.

MMP-9- and tPA-Mediated Reelin Processing.

Recently it was shown that the processing of Reelin bymetalloproteinase(s) is essential for normal cortical plate formation(Jossin, Y., and A. M. Goffinet. 2007. Reelin signals throughphosphatidylinositol 3-kinase and Akt to control cortical developmentand through mTor to regulate dendritic growth. Mol Cell Biol27:7113-7124), though the specific enzyme responsible remains as yetunknown. This discovery suggests that metalloproteinase-mediated Reelinprocessing may be important for directed Reelin signaling in the adultbrain as well. Both tPA and MMP-9 are candidate metalloproteinases withclearly demonstrated roles in regulating synaptic plasticity andcognitive function (Bozdagi, O., V. Nagy, K. T. Kwei, and G. W. Huntley.2007. In vivo roles for matrix metalloproteinase-9 in mature hippocampalsynaptic physiology and plasticity. J Neurophysiol 98:334-344; Nagy, V.,O. Bozdagi, A. Matynia, M. Balcerzyk, P. Okulski, J. Dzwonek, R. M.Costa, A. J. Silva, L. Kaczmarek, and G. W. Huntley. 2006. Matrixmetalloproteinase-9 is required for hippocampal late-phase long-termpotentiation and memory. J Neurosci 26:1923-1934; Huang, Y. Y., M. E.Bach, H. P. Lipp, M. Zhuo, D. P. Wolfer, R. D. Hawkins, L. Schoonjans,E. R. Kandel, J. M. Godfraind, R. Mulligan, D. Collen, and P. Carmeliet.1996. Mice lacking the gene encoding tissue-type plasminogen activatorshow a selective interference with late-phase longterm potentiation inboth Schaffer collateral and mossy fiber pathways. Proc Natl Acad SciUSA 93:8699-8704; Pang, P. T., and B. Lu. 2004. Regulation of late-phaseLTP and long-term memory in normal and aging hippocampus: role ofsecreted proteins tPA and BDNF. Ageing Res Rev 3:407-430; Zhuo, M., D.M. Holtzman, Y. Li, H. Osaka, J. DeMaro, M. Jacquin, and G. Bu. 2000.Role of tissue plasminogen activator receptor LRP in hippocampallong-term potentiation. J Neurosci 20:542-549; Baranes, D., D.Lederfein, Y. Y. Huang, M. Chen, C. H. Bailey, and E. R. Kandel. 1998.Tissue plasminogen activator contributes to the late phase of LTP and tosynaptic growth in the hippocampal mossy fiber pathway. Neuron21:813-825).

Reelin is processed by both tPA and MMP-9 to generate the major Reelinfragment products found in vivo (FIG. 12A-C, 12D-H). As it can be seen,tPA increases the 370 kDa (N-R6) and 80 kDa (R7-8) fragments under cellfree conditions (FIG. 12A-C), indicating that tPA cleaves Reelin betweenR6-R7 (FIG. 13). Cleavage of Reelin by Plasmin results in a spectrum ofproducts of previously unknown identity and specific retention of the180 kDa fragment. Application of recombinant tPA to primary neuronsresulted in a complete conversion of extracellular Reelin fromfull-length to the 370 and 180 kDa forms, and a decrease inintracellular 180 kDa Reelin. Furthermore, MMP-9 increases both the 370kDa (N-R6) and 180 kDa (N-R2) fragments, as well as a fragment foundjust below the well known 180 kDa fragment (FIG. 12D-H). These resultsunder cell free conditions support that MMP-9 can cleave Reelin at bothcleavage sites, R2-3 and R6-7; however, application of MMP-9 to primaryneurons led to a specific accumulation of the 180 kDa fragment in cellsand MMP-9 inhibition for 24 hours led to a dramatic increase infull-length cellular Reelin and decrease in cellular 180 kDa Reelin.These results suggest that under normal conditions, MMP-9 is responsiblefor cleaving Reelin between R2-R3 (See fragment map; FIG. 13). Takentogether, these preliminary data suggest that MMP-9 and tPA aresufficient for generation of the major Reelin fragments found in vivo.

As shown above, reelin protein processing in the hippocampus issusceptible to in vitro and in vivo synaptic activity. It also appearsthat MMP-9 and tPA are involved in the process of Reelin metabolism.Surprisingly, a single exogenous Reelin application enhances learningand memory for at least eleven days in adult wild-type mice. Whenconsidering the role of lipoprotein receptors in Aβ clearance, and theidentification of Reelin association to Aβ plaques in an AD mouse model,the question of the role of Reelin in the etiology and pathogenesis ofAD becomes a timely and important area of research. Moreover, the nowimproved understanding of the mechanisms and implications of Reelinprocessing provides, inter alia, AD therapeutic interventions aimedtoward removal of Aβ and improvement of cognitive function.

Moreover, all that is known regarding Reelin localization in the adultbrain has been generated using an antibody that recognizes the N-R2region. The N-R2 region is present in the full-length (N-R8), N-R2 andN-R6 fragments of Reelin, but not in the other major fragments.Therefore, the 3-epitope mapping approach ((FIG. 13) affordsunprecedented spatial resolution to monitor changes in Reelin productproduction and localization.

In order to characterize specific fragments produced by tPA- andMMP-9-dependent Reelin processing in the context of normal synapticfunction and memory formation, the inventors generatedcleavage-resistant Reelin mutant constructs using site-directedmutagenesis FIG. 14). Reelin mutants include constructs resistant tocleavage (Rln-Res) by tPA at R2-3, to MMP-9 at R6-7 and to both enzymesat R2-3 and R6-7. Fragments mimicking cleavage by tPA or MMP-9 with, orwithout a cleavage resistant site are also contemplated. Onecomplementary Reelin construct is tagged in an identical fashion as theRln-Res protein; however, it does not contain the two altered sites forcleavage (Reelin cleavage labile; FIG. 14)). A tagged fragment producedwith both sites mutated (negative control construct) and a tagged R3-6fragment shown to bind ApoER2 and VLDLR (potential positive control) isincluded. The Reelin constructs are sub-cloned into mammalian expressionvectors containing N-terminal polyhistidine tags and/or C-terminal Myctags to allow later recognition of exogenous Reelin. The exact cleavagesites can be identified by using purified full-length Reelin reactedwith either tPA or MMP-9 therefore the resultant fragments can beisolated.

Reelin Application Recovers Spine Density in HRM and Reelin-Null Mice

In cultured hippocampal neurons, reelin signaling is required for normaldevelopment of dendritic structures. In the absence of reelin or theintracellular adaptor protein Dab1, neurons exhibit stunted dendriticgrowth and a reduction in dendritic branches, a phenotype analogous tothat seen in neurons lacking the reelin receptors apoER2 and VLDLR (NiuS, Renfro A, Quattrocchi C C, Sheldon M, D'Arcangelo G. Reelin promoteshippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway.Neuron 2004; 41:71-84). The HRM exhibits a deficit inhippocampal-dependent contextual fear conditioned learning and synapticplasticity in area CA1 of the hippocampus. It is believed that thesebehavioral and physiologic phenotypes of the HRM are due in part toreduced or inhibited synaptic connectivity. This is supported by theobservation that HRM have a reduction in spine density (FIG. 15).

Dendritic spines are small protrusions that cover the surface ofdendrites and bear the postsynaptic structures that form excitatorysynapses. Abnormal shapes or reduced numbers of dendritic spines arefound in a number of cognitive diseases, such as Fragile X syndrome,William's syndrome, Rett syndrome, Down's syndrome, Angelman syndromeand autism. A reduction in the number of dendritic spines suggests thata constitutive level of reelin/lipoprotein receptor-mediated signalingis required for development of dendritic structures, which are crucialfor intensive information processing by the neurons. This notion is inagreement with studies showing that heterozygote reeler mice exhibitreduced dendritic spine densities and impaired performance in certainlearning and memory behaviors.

Hippocampal neurons cultured from reeler embryos had significantly lessdendritic spines, a phenotype that can be rescued by addition ofexogenous recombinant reelin to the culture. Organotypic hippocampuscultures were created from 6-7 day-old wild-type, HRM andReelin-deficient mice and treated with 5 nm Reelin for 21 days.Fluorescent dye was injected into neuronal cells by administering wholecell patch clamp current and the cells were visualized under theconfocal microscope after fixation. Reelin-treated of HRM cells showedan increase in dendritic spine density after 21 days compared to agematched neurons from wild-type culture (FIG. 15B). In contrast, mock(conditioned media from non-stably transfected cells) application showedno change in spine density (FIG. 15C). The same experiment in reelinknockout mice showed that reelin application also rescued the dendriticspine density compared to mock controls (FIGS. 15C and 15F). Both thereelin treated cells resembled the dendritic spine morphology seen in WTcells (FIG. 15D) and when quantified, dendritic spines significantlyincreased in reelin-treated HRM cultures compared to mock treatedcontrols and are similar to spine density levels observed in wild-typecultures (FIG. 15A).

Treatment of organotypic cultures consisted of repeated 5 nM Reelinapplication every 3 days for 21 days. To verify that this applicationprotocol represented a chronic application of reelin, and reelin was notbeing degraded or actively removed from the media, the inventors removed15 ul of media from culture plates at times of 0, 6, 12, 24, 48, 72, and96 hours following reelin application. Western analysis of thesealiquots showed no degradation or reduction in Reelin (FIG. 15G). Thus,the increase in spine density is due to reelin present at physiologicrelevant levels for the entire 21 day application.

In Vivo Reelin Application Effects on Overall Behavioral Responses.

Mice lacking reelin exhibit abnormal lamination of neuronal layers,which is most severely seen in the cortex, cerebellum, and hippocampus.The Reelin knockout exhibits the “reeler” phenotype, characterized byrest tremor and ataxia. Although the Cajal-Retzius cells eventuallydegenerate after the completion of development, reelin continues to beexpressed by GABAergic interneurons in the cortex and hippocampus. Inthe adult, as in the developing brain, Reelin's molecular effects aremediated through two receptors: the very low density lipoproteinreceptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2).Reelin-dependent signaling through ApoER2 and VLDLR occurs throughhetero- or homo-dimerization of receptors and can activate the CDK-5 andPI3-K signal transduction pathways. Reelin signaling is also linked tomodulation of synaptic plasticity and memory formation.

The heterozygote reelin mouse (HRM) exhibits haploinsufficiency and a50% reduction in reelin protein levels, but does not lead to an overt“reeler” phenotype. Instead, Reelin haploinsufficiency manifests as verysubtle neuroanatomical, physiologic and behavioral deficits. Theseinclude a decrease in dendritic spines in the parietal-frontal cortex(PFC) pyramidal neurons in addition to basal dendritic cells ofhippocampal CA1 pyramidal neurons and cortical neuropil hypoplasia. TheHRM displays a reduced density of nicotinamide-adenine dinucleotidephosphate-diaphorase (NADPH-d)-positive neurons in the cortical graymatter, altered dopaminergic markers in the mesotelencephalic dopaminepathway. The HRM shows impaired short-term and long-term plasticity inhippocampal CA1 synapses. Long-term potentiation (LTP) is disruptedusing both high frequency stimulation and pairing stimulation protocols.Behaviorally, the HRM exhibits an age-dependent decrease in prepulseinhibition.

The HRM has often been referred to as a possible mouse model for humanschizophrenia. Reelin mRNA and protein levels are reduced in post-mortembrains of schizophrenic patients resulting in approximately 50% of thatfound in normal control post mortem brains. Investigation of the Reelerheterozygote found other similarities to the human condition, including:decreased GAD67 expression, decreased tactile and acoustic prepulseinhibition, and reduced spine density. Schizophrenia is also associatedwith severe cognitive impairment and disordered thinking. This manifestsas a lack of overall attention, impairment of information processingdisrupting both declarative and nondeclarative memories. Importantly,HRM show a similar cognitive dysfunction, observed as reducedassociative fear conditioned learning.

An HRM breeding pair (B6C3Fea/a-Reln^(rl/+ strain) was obtained from the Jackson Laboratory. The offspring of both HRM were genotyped by using genomic DNA from a)2 mm diameter earpunch. The primer sequences were, forward:5′-taatctgtcctcactctgcc-3′ (SEQ ID NO:1); reverse:5′-acagttgacataccttaatc-3′ (SEQ ID NO:2); reverse mutated:5′-tgcattaatgtgcagtgttgtc-3′(SEQ ID NO:3). Animal care and use protocolwas approved by the Institutional Animal Care and Use Committee ofVanderbilt University.

The culmination of research on Reelin's actions in developing CNS andadult cognitive processes raises the question of whether the cognitivedeficits in HRM are due to reelin haploinsufficiency, leading to adecrease in signal transduction and LTP formation, or reduction indendritic spines, resulting in decreased information processing andstorage in areas involved in learning and memory. Alternatively, HRMshow reduced spine density, thus, these deficits may be due todevelopmental defects that result in the mis-wiring of critical regionsof the CNS.

The increase in sEPSCs in wild-type mice, but not in Reelin knockoutmice despite chronic reelin exposure indicated that spine formation wasnot the sole factor influencing spontaneous synaptic activity incultured neurons. This would suggest that developmental abnormalitiesresulting in altered synaptic connectivity in the hippocampus of HRM,and to a greater extent in the Reelin-deficient mice, were theunderlying basis for the cognitive deficits in the HRM.

The HRM and wild-type mice are similar for open field and elevated plusmaze. These behavioral tests are essential for evaluation and properdetermination of associative fear conditioning results. In addition, theopen field and elevated plus maze tasks allow assessment of anydifferences in locomotor activity or anxiety after the cannulationplacement and injection.

For the open field tests, general locomotor activity was measured usingthe open field task. Animals were placed in the open field (27×27 cm)chamber for 15 min in standard room-lighting conditions. Activity in theopen field was monitored by 16 photoreceptor beams on each side of thechamber and analyzed by a computer-operated (Med Associates) animalactivity system.

For the elevated plus maze experiments, mice were placed in the elevatedplus maze one hour after they had completed the open field task to testtheir levels of anxiety. The apparatus consisted of two opposing openarms (30 cm×5 cm) and two opposing closed arms (30 cm×5 cm×15 cm)connected by a central square platform and was 40 cm above the ground.Testing took place under standard-lighting conditions. Mice were placedin the open arms facing the closed arms at the beginning of the 5 minutesession. The number of entries and the total time spent in the open armswere recorded.

Bilateral intracerebroventricular cannulations on HRM mice were followedby evaluating open field and elevated plus maze tests. Following a 5 dayrecovery period from the surgical procedure, these mice were injectedwith 1 ul of either mock or reelin through two PE50 tubes attached totwo Hamilton syringes. Mice were visually assessed daily for overallhealth following surgery. All mice used for these studies showed nosigns of infection assessed by visual inspection of the site of incisionand rectal temperature monitored daily.

The injection of 1 ul of a concentrated Reelin solution represented afinal distributed concentration of 5 nM. To test for dispersion ofReelin cannulated wild type mice were injected monolateral with 1 ulReelin and sacrificed 1 hour following injection. Brains were fixed andimmunohistochemical analysis for Reelin was performed. No discernabledistribution of Reelin was seen in the Reelin injected hemisphere,however, an increase in overall Reelin immunoreactivity was observed inthe treated versus non treated hemispheres. This suggests that Reelinquickly diffuses from the ventricle by the time of behavioral testing.

One hour after Reelin injection the experimental mice were placed in theopen field chamber and distance traveled over a 15 minute period wasmeasured. Immediately following the open field task, mice were placed inthe elevated plus maze and the number of entries and percent time in theopen arms were measured. A greater amount of time spent in the closedarms compared to the open arms is an index of higher anxiety. Nodifferences were seen between the mock and reelin treated heterozygotemice in these two tasks (FIG. 16A-C).

Mice normally exhibit a startle response to loud noise but if a moderatenoise is presented prior to the loud noise, the startle response isattenuated, an effect known as prepulse inhibition (PPI). PPI representsanother compelling behavioral phenotype of the HRM that recapitulateshuman schizophrenia. PPI was performed one hour after elevated plusmaze. The mouse was placed in a Plexiglas cylinder in a dark PPI chamber(Med Associated Inc.; St. Albans, Vt.) with the presence of backgroundnoise provided by a fan. After mice were allowed to acclimate for 5minutes in the chamber, they were underwent a random presentation offive stimulus trial types: 120 db stimulus startle alone, and each of a70, 76, 82, and 88 db prepulse followed by a 120 db startle for a totalof 9 trials per type. The percent prepulse inhibition and the peakstartle were measured using the Startle Reflex 5 software.

The inventors have previously shown that the HRM show a deficit in PPI,specifically at the 82 dB prepulse. To determine whether Reelin rescuesthis deficit, the inventors performed PPI in Reelin-injected cannulatedmice. Following the elevated plus maze, mice were placed in the startlereflex chamber and given a random presentation of 5 trial types: noprepulse with a 120 dB acoustic startle, or 70, 76, 82, 88 dB prepulseswith a 120 dB acoustic startle. The inventors saw that there was nodifference in the startle to acoustic stimulation, where no prepulse waspresented with a 120 dB acoustic startle between the mock treated andreelin treated HRM (FIG. 16E). Additionally, there was no difference inthe PPI between both treatment groups at any of the prepulse levels(FIG. 16D).

The HRM shows a deficit in associative learning when compared to theirwild-type littermates. Contextual fear conditioning was performed onReelin and Mock-treated HRM at 5 hours post-injection to assess whetherReelin haploinsufficiency is responsible for this change rather thanpermanent developmental defects. Fear conditioning was performed 2 hoursafter PPI. The conditioning chamber (26×22×18 cm; San Diego Instruments,San Diego, Calif.) was made of Plexiglas and was equipped with a gridfloor for delivery of the unconditioned stimulus (US) and photobeams tomonitor activity. The conditioning chamber was placed inside asoundproof isolation cubicle.

Training occurred in the presence of white light and background noisegenerated by a small fan. Each mouse was placed inside the conditioningchamber for 2 minutes before the onset of a conditioned stimulus (CS),an 85 dB tone, which lasted for 30 seconds. A 2 sec US foot-shock (0.5mA) was delivered immediately after the termination of the CS. Eachmouse remained in the chamber for an additional 60 seconds, followed byanother CS-US pairing. Each mouse was returned to its home cage afteranother 30 seconds. The test for contextual fear memory was performed 1,24, and 72 hours after training by measuring freezing behavior during a3 minute test in the conditioning chamber.

Freezing was defined as lack of movement in each 2 second interval. Cuedfear memory was tested in the presence of red light, vanilla odor, andthe absence of background noise. The grid floor was covered and thewalls were covered with alternating black and white plastic panels. Eachmouse was placed into this novel context for 3 minutes at 1 hour and 24hours after training. They were exposed to the CS for another 30 minfollowing this. Freezing behavior was recorded and processed by the SDIPhotobeam Activity System software throughout each testing session.

The aversive unconditioned stimulus (US), a 5 mA foot-shock, was pairedtwice with an auditory tone (conditioned stimulus, CS). During thetraining period, both animals showed similar levels of freezing afterthe presentation of the US with an increasing trend of freezing (FIG.17A). This indicates that the acquisition of the fear memory is similarin both groups and freezing ability is similar. Mice were placed backinto the context in which they were trained at 1, 24 or 72 hoursfollowing training. There was no difference between the Reelin treatedmice compared to the mock treated controls at 1 hour post-training (FIG.17B). However, 24 hours after training, mice were placed into thechamber for the second time to examine the effects of Reelin onlong-term memory formation. Reelin-treated mice showed a significantincrease in percent freezing compared to mock-treated controls (FIG.17C). These levels are similar to the levels of freezing in WT mice thatthe inventors have previously shown (˜70%), while the mock-treatedcontrols resembled our HRM. This suggests that reelin rescues thehippocampal-dependent associative learning deficits seen in the HRM toresemble the WT mouse. When tested 72 hour following training, there isno statistical significance between the two treatment groups, althoughthere is trend for an increase in freezing in the Reelin group comparedto mock controls (FIG. 17D). This may represent a consolidation effectin some of the mice in that the re-introduction into the context in theabsence of the aversive stimulus may lead to the recall andre-organization of the memory. The ensure that both treatment groups hadsimilar sensitivities to the foot-shock, a shock threshold test wasperformed. No difference was seen between reelin-treated andmock-treated mice (FIG. 17E). Thus, Reelin replacement to the CNS of HRMrescues the contextual fear conditioning defect.

In Vivo Application of Receptor Associated Protein.

The results above show that increasing Reelin, and subsequent Reelinsignaling, in the hippocampus rescues the cognitive deficit. If thedecrease in ApoER2 and VLDLR signaling is responsible for the cognitivedefects in HRM, then one should be able to mimic these behavioralchanges by blocking ApoER2 and VLDLR. Receptor Associated Protein (RAP)serves as a molecular chaperone for the family of lipoprotein receptorsallowing transport to the plasma membrane without premature binding toligand. Applied exogenously, RAP binds to the extracellular portion ofthe lipoprotein receptor and acts as an effective antagonist. Exogenousapplication of RAP results in association of inserted receptors and caneffectively block extracellular ligand-induced signaling. The use of RAPas a biological antagonist has previously been used to blockreceptor-induced signaling in culture and tissue, and can effectivelyblock long-term potentiation (LTP) in wild-type hippocampus. Thus, thebehavioral tests were performed on wild-type mice injected with 1 ul ofconcentrated GST-RAP or GST as a negative control.

Exogenous GST-RAP or GST (used as a negative control) ha no effect onoverall behavior (FIGS. 18A and B-C) and no change was seen in PPI oracoustic startle (FIG. 18D-E). There were no changes in freezing duringfear condition experiments or in testing to the context 1 hour aftertraining (FIG. 19A-C). However, GST-RAP injection resulted in asignificant decrease in freezing to the context to a level identical tothat seen in our HRM Mock treated animals and those levels previouslyreported in HRM mice (FIG. 19C). No differences were seen in shockthresholds between the two treatment groups (FIG. 19D).

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention.

What is claimed is:
 1. A method of treating monogenic disorders,neuro-psychiatric disorders, neuro-traumatic disorders, orneuro-degenerative disorders, comprising: administering atherapeutically effective amount of a Reelin fragment to a subject;wherein the Reelin fragment is a 450 kDa Reelin protein fragment, a 370kDa Reelin protein fragment, a 180 kDa Reeling protein fragment, or acombination thereof to the subject; wherein the monogenic disorders arecharacterized by cognitive impairment; and wherein theneuro-degenerative disorders are characterized by cognitive impairment.2. The method of claim 1, wherein the therapeutically effective amountof the Reelin fragment is about 5 nM.
 3. The method of claim 1, whereinthe disease or disorder of the nervous system is selected from the groupconsisting of fragile X syndrome, William's syndrome, Rett syndrome,Down's syndrome, Angelman syndrome, autism, ischemia, hypoxia,Alzheimer's disease, Reelin deficiency, schizophrenia and stroke.
 4. Themethod of claim 3, wherein the therapeutically effective amount ofReelin or an agonist of a lipoprotein receptor is about 5 nM.
 5. Themethod of claim 1, wherein the symptom of a disease or disorder of thenervous system is selected from the group consisting of a deficiency indendritic spine density, diminished long-term potentiation, diminishedsynaptic plasticity and associative learning deficits.
 6. The method ofclaim 5, wherein the therapeutically effective amount of Reelin or anagonist of a lipoprotein receptor is about 5 nM.